Fluorescence imaging provides a powerful tool for identifying and characterizing selected contents of assay mediums, particularly biological assays containing in vivo or in vitro contents of interest. Specific molecular structures, including individual proteins or nucleic acids, and cell types or tissues in the form of single or array-based assay mediums can be targeted to fluoresce for identification or characterization.
Within typical fluorescence spectroscopic instruments, the assay mediums are (a) irradiated at predetermined wavelengths that excite fluorophores associated with the selected contents and (b) imaged onto detector arrays for capturing the resulting Stokes-shifted luminescence. Information concerning the spectral content, polarization, intensity, lifetime, distribution, and shape of the luminescent targets over spatial and temporal dimensions can be extracted for identifying or characterizing the selected contents.
Systematic errors arise from spatial non-uniformities associated with both the irradiation and imaging of the assay mediums. For example, two-dimensional assays generally require uniform irradiance over the field of view of the assay medium. The requirements for uniform irradiance can include not only uniform intensity over the field but also uniform spectral content, polarization, angular profile (field pattern), and dose (integrated flux) throughout the field. Systematic errors can arise as field anisotropies among these requirements for uniform irradiance. Systematic errors associated with imaging include optical aberrations such as field curvature, distortion, and chromatic aberration, as well as detection errors such as absorbance errors, dark field error, and variations in detection efficiency. Stray radiation can also influence baseline values.
Calibration techniques for the fluorescence spectroscopic instruments have been applied to quantify and compensate for non-uniformities and other systematic errors associated with the irradiation and imaging of the assays. For example, reference plates containing fluorescent material uniformly distributed throughout the plates have been irradiated and imaged in place of the assay mediums to detect anisotropies or other deviations from expectations. The reference plates occupy the same or an enlarged area of the field of view as the array mediums intended for evaluation and respond to incident light in the same way throughout the occupied area of the field. Any spatial deviations in the fluorescence response, i.e., the fluorescent emission of light at Stokes-shifted wavelengths, are attributable to disparities in the irradiation. The fluorescence response itself is subject to further variation as imaged onto a detector array. The end result is one or more digital images within which deviations from a norm are indicative of errors or other anomalies of the irradiation and imaging systems.
The information acquired from reference plates can be used to (a) correct or otherwise adjust the irradiation and illumination systems, (b) scale the results to a known standard, or (c) provide a baseline for distinguishing systematic variations in the results from true differences within sample assays. For example, fluorescent images of the sample assay mediums can be normalized to fluorescent images of the reference plates having predefined responses.
Many applications require the simultaneous study of different contents within the assay mediums, such as multiple proteins or protein states (e.g., phosphorylation). Unique fluorescent tags that emit different wavelengths upon excitation are associated with the different contents. Different wavelength emitting fluorescent tags excited by the same wavelength are particularly useful for comparing the contents simultaneously. However, different wavelength emitting fluorescent tags excited by different wavelengths provide more flexibility for separate analyses of the contents.
Known reference plates for calibrating fluorescence spectroscopic instruments are generally formed by a coated substrate, a gel plate, or solid film containing one or more fluorescent agents. Some such plates have included multiple fluorescent agents, which can differ depending upon the particular fluorescent tags or probes intended for use within the assay medium. However, combinations of fluorescent agents with overlapping excitation and emission wavelengths can produce interactions that obscure the individual contributions of fluorescent agents. The wavelengths emitted by a first fluorescent agent can be absorbed and hence excite a second fluorescent agent, which diminishes the contribution of the first fluorescent agent and amplifies the contribution of the second fluorescent agent.